Composition of matter and process

ABSTRACT

New antibiotics, N-demethyl-7-0-demethylcelesticetin (IV) and Ndemethylcelesticetin (IVa), are produced concomitantly by the controlled fermentation of the new microorganism Streptomyces caelestis strain 22218a in an aqueous nutrient medium. These antibiotics are antibacterially active and also can be converted to various antibacterially active analogues.

United States Patent [191 Argoudelis et al.

[ Nov. 12, 1974 COMPOSITION OF MATTER AND PROCESS [75] Inventors: Alexander D. Argoudelis, Portage;

John H. Coats, Kalamazoo, both of Mich.

[73] Assignee: The Upjohn Company, Kalamazoo,

Mich.

[221 Filed: Dec. 13, 1973 [21] Appl. No.: 424,623

Related US. Application Data [62] Division of Ser. No. 261,724, June 12, 1972.

[52] US. Cl 195/80 R [51] Int. Cl C12d 9/00 [58] Field of Search 195/80 R [56] References Cited UNITED STATES PATENTS 2,928,844 3/1960 Boer et al. 195/80 R Primary Examiner-A. Louis Monacell Assistant ExaminerRobert J. Warden Attorney, Agent, or FirmRoman Saliwanchik [5 7 ABSTRACT 5 Claims, No Drawings 3,847,746 1 2 COMPOSITION OF MATTER AND PROCESS celesticetin (U-40,585), are obtained by culturing H a Streptomyces caclcstis strain 222.1 8a, NRRL 5481, in CROSS RH: ERENU: IO RELA PhD APPLICATION an aqueous nutrient medium. NDemethylJ-O-demethylcelestricetin can be represented by the following This is a division of co-pending application Ser. No. t u t ral for ul 26l 724, filed on June 12, 1972.

BACKGROUND OF THE INVENTION Celesticetin is an antibacterially active compound which is disclosed in US. Pat. No. 2,928,844. It can be It) represented by the following structural formula:

0 on ll SCH2 CH20C N-Demethylcelesticetin can be represented by the following structural fonnula: 0H 0 Celesticetin can be hydrolyzed according to the pro- H CH8 cess disclosed in US. Pat. No. 2,851,463 to produce 1 the compound desalicetin which can be represented by N the following structural formula: CH O-CH H lVa CH. CH 3 a r --N CH g CH O-CH g 0 I H H t H Y i l I OH H I C 'N,--,-CH H SCHZCHQOC H tscH cH oH Upon the treatment of N-demethyl-7-0-demethylcelesticetin (IV) with a suitable base. for example, so-

5 dium hydroxide, potassium hydroxide. ammonium hydroxide, and the like, there is produced N-demethyl-7- O-demethyldesalicetin which can be represented by the following structural formula:

These celesticetin compounds are structurally re lated somewhat to the well-known anitibiotic lincomycin which can be represented by the following structural formula:

it CH5 CH3 CH3 H N I l HO-CH v HO-CH H CSH? H fl N CH .ll HO H 0 HO 0 H 0H OH H H SCHQCHQOH H SCHs H OH H OH , ,5 Likewise, upon treatment of N-demethylcelesticetin BRIEF SUMMARY OF THEINVENTIQN (lVa) with a base, as above, there is produced h novel Compounds fth invention, N dmethy| N-demethyl-desalicetin which can be represented by 7-0-demethylcelesticetin u-39,579) and N-demethylthe following Structural formula:

H cH I u CHa-CH H l (Ii-"N CH VB 0 H0 0 H M H H SCHz CHzOH H OH Treatment of N-demethyl-7-O-demethylcelesticetin with hydrazine hydrate yields B-hydroxyethylthiolincosaminide (B-HETL) which can be represented by the following structural formula:

l HO-(IIH H2N-CH Vl OH H 5 CH2 CH2 OH ln addition to B-hydroxyethylthiolincosaminide, there is produced L-proline hydrazide which can be transformed to L-proline hydrochloride by heating with aqueous hydrochloric acid. The sequence of this latter reaction can be shown as follows:

HCl

N coHHHH,

1 coca H N-Demethyl-7-O-demethylcelesticetin (N) can be converted to various antibaeterially active analogues. Compound (IV) and these analogues can be represented by the following structural formula:

T CH3 N +x 0 H v fi cH 0 HO 0 ll H OH HQCHQCHQOC /m H 0R OH wherein X is OH in the (R) and (S) configuration; halo in the (R) and (S) configuration: alkoxy of not more than carbon atoms in the (R) and (S) configuration excepting where X is (R)OCH;,. R is hydrogen and R, is methyl; R is hydrogen or is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms. or a halo-. nitrohydroxy-, amino, thiocyano-, and lower alkoxyhydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive, and

and R is hydrogen, alkyl of from one to 20 carbon atoms, inclusive, or hydroxyalkyl of from two to five carbon atoms, inclusive.

N-Demethylcelesticetin (lVa) can be converted to various antibacterially active analogues. These analogues can be represented by the following structural wherein R is hydrogen or is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxyamino-, thiocyano-, and lower alkoxy-hydrocarbon carboxylic acid acyl of not more that 18 carbon atoms. inclusive,

and R is hydrogen, or alkyl of from two to 20 carbon atoms, inclusive, or hydroxyalkyl of from two to live carbon atoms. inclusive.

N-Demethyl-7-()-demcthyldesalicetain (V) can be converted to various antibacterially active analogues. Compound (V) and these analogues can be represented by the following structural formula:

T17 CH3 H w N CH I 0 HO 0 H H H' SCHzCHgOR wherein X, R, and R are as defined above; and R is hydrogen or is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydrxy-, amino-, thiocyano-, and loweralkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive, except that R is not the salicylic acid moiety when X is (R)-OCH and R is hydrogen and R is methyl.

N-Demethyldesalicetin (Va) can .be converted to various antibacterially active analogues. These analogues can be represented by the following structural formula:

wherein R and R are as defined above; and R is hydrogen or is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thiocyano-, and lower-alkoxyhydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive; except that R is not the salicylic acid moiety when R is hydrogen and R is methyl.

B-Hydroxyethylthiolincosaminide (V1) can be converted to various analogues which can be represented by the following structural formula:

loxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, and eicosyloxy and the isomeric forms thereof. Examples of halo are chlorine, bromine and iodine.

Examples of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxyamino-, thio-, cyano-, and loweralkoxyhycrocarbon carboxylic acid acyl of not more than 18 carbon atoms. inclusive, are as disclosed in US. Pat. No. 3,426,012, column 5, line 64 to column 6, line 47. Examples of alkyl of from one to 20 carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, dccyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and eicosyl, and the isomeric forms thereof.

DETAILED DESCRIPTION CHEMICAL AND PHYSICAL PROPERTIES OF N- DEMETHYL-7-O-DEMETHYLCELESTICETIN HYDROCHLORIDE Specific rotation:

[ 111 =+122 (C, 1, water) =+163 (c. l. dimethylformamide) Ultraviolet Absorption Spectra:

In water:

In 0.01 N aqueous NaOH:

A max. at 204 m,u(21=66.1)l15=33,001l) A max. at 238 mu (a= 17.66: e=8,85()) A max. at 303 my. (a 7.20;e=3.600) A max. at 241 m (a =11.84; e=5.900)

A max. at 331 mp. (a=8.39; e=4,200)

wherein X, R. and R are as defined above.

Compounds of formula IX can be converted to an- .tibacterially active compounds by coupling with a pyrrolidine-carboxylic acid as disclosed in US. Pat. No. 3,514,440. The compounds formed, as well as the process, are disclosed in said patent.

Examples of alkoxy of not more than 20 carbon atoms are methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decy- Infrared Absorption Spectra: Following is a tabulation of the infrared absorption spectrum wave lengths in mineral oil mull expressed in reciprocal centimeters:

Band Frequency Band Frequency Following is a tabulation of the infrared absorption spectrum wave lengths where the compound is pressed into a KBr pellet expressed in reciprocal centimeters:

Band Frequency Band Frequency Infrared band intensities, throughout this disclosure, are indicated as S, M, and W, respectively, and are approximated in terms of the backgrounds in the vicinity of the bands. An S band is of the same order of intensity as the strongest in the spectrum; M bands are between 1/3 and 2/3 as intense as the strongest band; and, W

bands are less than l/3 as intense as the strongest band.

These estimates are made on the basis of a percent transmission scale. SH denotes a shoulder.

ANTIBACTERIAL ACTIVITY OF N-DEMETHYL-7-0-DEMETHYLCELESTICETIN Minimum Inhibitory Con- TEST MICROORGANISM centration in mcq./ml.

Staphylococcus aureus 15.6 Streptococcus hemolyticus 7.8 Streptococcus faecalis 31.2 Escherichia coli 500 Proteus vulgaris 500 Klcbsiella pneumoniae 500 Pseudomonas aeruginosa 500 The above antibacterial spectrum was obtained by a tube dilution assay procedure. This procedure was conducted with the medium BHI (Brain Heart Infusion Broth, Difco, Detroit, Michigan). Assay tubes 13 mm. X 100 mm.) were prepared in the customary manner as set out in Snell, E. E., Vitamin Methods, Vol. 1, Academic Press, Inc., New York (1950), page 327. Test organisms grown for 18 hours at 37C. were added to inoculate the test medium. The results were read at 17 hours.

CHEMICAL AND PHYSICAL PROPERTIES OF N-DEMETHYLCELESTICETIN HYDROCHLORIDE Elemental Analysis:

Elemental analysis showed the presence of C, H, N, S and CI in the molecule. High resolution mass spectrometry on a trimethylsilyl ether derivative showed molecular ion at 874.3967 mass units (average of 4 de' terminations). Theoretical for C H- N O SSi is 874.3958 mass units. These results indicate the molecular formula of C H N O S for the antibiotic.

Furthermore the M"-CH peak was measured at 859.3724.

Theoretical for C H N O SSi is 859.3723. Molecular Weight: 514 (determined by mass spectrometry). Specific Rotation: [a] l l2.5(c, 1, water). Ultraviolet Absorption Spectrum (in methanol):

It max. at 238 mp. (a= 15.5)

kmax. at 304 mg (a 7.2) Infrared Absorption Spectra: Following-is a tabulation of the infrared absorption spectrum wave lengths in mineral oil mull expressed in reciprocal centimeters:

Band Frequency Band Frequency (cm Intensity (cm") Intensity 3300 S 1 I55 S 3080 S I138 S 2925 (oil) S I086 S 2860 (oil) S I057 S I670 S I002 M I610 S 986 M I579 S 975 M I562 S 950 W I483 S 905 W I467 (oil) S 865 M I455 (oil) S 815 W I375 (oil) 5 797 M I322 S 757 S I296 S 719 (oil) M I247 S I S I210 S Following is a tabulation of the infrared absorption spectrum wave lengths where the compound is pressed into a KBr pellet expressed in reciprocal centimeters:

Band Frequency Band Frequency (cm) Intensity (cm") Intensity 3380 8 I247 S 3260 S I2 I 2 S 3080 S I I55 5 2980 S I I37 S 2930 M I085 S 2820 M I055 S I668 S 985 M 16 I 0 S 950 M 1578 M 905 W 1557 S 867 M 1486 S 8 I 2 W I455 M 796 M I387 S 755 M I320 S 700 S ANTIBACTERIAL ACTIVITY OF N-DEMETHYLCELESTICETIN Minimum Inhibitory Con- TEST MICROORGANISM centration in mcq./ml.

Staphylococcus aureus I5.6 Streptococcus hemolyticus 0.I25 Streptococcus faecalis 7.8 Escherichia coli 500 Proteus vulgaris 500 Klebsiella pneumoniae 250 Pseudomonas aeruginosa 500 Diplococcus penumoniae 0.5

The above test results were obtained using the assay and conditions disclosed above for the antibacterial test of N-demethyl-7-O-demethylcelesticetin.

THE MICROORGANISM The microorganism used for the production of the antibiotics of the subject invention is a mutant of Streptomyces caelestis, 2418, which has been named Streptomycete Streptomyces caelestis strain 2221811. This mutant is distinguishable from Streptomyces caelestis taxonomically, as shown hereinafter, and by its ability to produce N-demethyl-7-O-demethylcelesticetin and N-demethylcelesticetin. A subculture of the living organism can be obtained from the permanent collection of the Northern Utilization and Research Division, Agricultural Research Service, US. Department of Agriculture, Peoria, Illinois, USA. Its accession number in this repository is NRRL 5481.

The microorganism of this invention was studied and characterized by Alma Dietz of the Upjohn Research Laboratories.

DESCRIPTION OF THE MICROORGANISM Streptomyces caelestis strain 222l8a, UC 5515.

Streptomyces caelestis strain 222l8a is compared with the parent culture (which is also the type culture) Streptomyces caelestis [DeBoer, C., A. Dietz, J. R. Wilkins, C. N. Lewis and G. M. Savage. l954--1'955. Celesticetin A new, crystalline antibiotic. l. Biologic studies of celesticetin. Antibiotics Annual, New York. Medical Encyclopedia, Inc., 1955. pp 831-836], UC 2011, NRRL 2418. Taxonomic Methods: The methods used were those cited in Dietz [Dietz, A., 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol. 94:2022-2026] and, in part, those cited in Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb, 1966. Methods for characterization of Streptomyces species. iii. J.

Syst. Bacteriol. 162313-340]. Color Characteristics: Aerial growth blue (sporulating) for the parent; white with trace blue or some media (non-sporulating to trace sporulating) for the mutant. Parent: melaninpositive; strain: melanin-negative. Appearance on Ektachrome [Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. NY. Acad. Sci. 602152-154] is given in Taable 1. Reference color characteristics are given in Table 2. The parent culture may be placed in the White (W), Green (GN), or Gray (GY) series of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1962. System of color wheels for taxonomy. Appl. Microbiol. 11:335-338]; the mutant in the White (W) and Yellow (Y) series. Microscopic characteristics: The parent and the mutant have open spiral to spiral sporophores (in the sense of Pridham et al., [Pridham, T. G., C. W. I-Iesseltine, and R. G. Benedict. 1958. A guide for the classification of Streptomycetes according to selected groups. Placement of strains in morphological sections. Appl. Microbiol. 6:52-79]) bearing rectangular smooth spores (in the sense of Dietz and Mathews [Dietz, A., and J. Mathews. 1970. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21:527-533]) with ridged surface structure. The mutant strain has distorted mycelium with large round bodies when observed by the transmission electron microscope. Cultural characteristics: See Table} Carbon utilization: See Tables 4 and 5. Temperature: S. caelestis strain 2221811 and the parent S. caelestis grow at 18 C. Growth is poor at the extremes and optimum at 28 37C. Antibiotic Producing Properties: Streptomyces caelestis strain 22218a produces 7- O-demethyl-N-demethylcelesticetin (U-39,579) and N-demethylcelesticetin (U-40,5

DISCUSSION Streptomyces caelestis strain 222l8a, NRRL 5481, a mutant of Streptomyces caelestis UC 201 1, NRRL 2418, is shown to have cultural characteristics distinctly different from those of the parent. It does have the distinctive sporophore and spore characteristics of the parent strain in its sporulating sectors which are celestial blue (the color of the parents sporulating aerial.

growth).

The culture characterized as Streptomyces caelestis strain 22218a has been so designated in accordance with Recommendation 8a of the International Code of Nomenclature of Bacteria, 1966, edited by the Editorial Board of the Judicial Commission of the lntemational Committee on Nomenclature of Bacteria. Intern. J. System. Bacteriol. 16: 459-490, in which a number which is a laboratory distinguishing mark may be used to designate the descendents of a single isolation in pure culture.

The characteristics of Streptomyces caelestis strain 222l8a, NRRL 5481, are given in the following tables:

Table 1 Appearance of S. caelestis Cultures on Ektachrom Table 2 Reference Color Characteristics of S. caelestis Cultures Table 3 Cultural Characteristics of S. caelestis Cultures Table 4 Utilization of Carbon Compounds in the Synthetic Medium of Pridham and Gottlieb Table 5 Utilization of Carbon Compounds in the Synthetic Medium of Shirling and Gottlieb TABLE 1 Apgurgnce of S. caelestis Cultures on Ektachrome I S. caelestis S. caelestis Agar medium TABLE 2 Reference Color Characteristics of S. caelestis Cultures Color flarmony Manual 3rd ed.. 1948* NBS Circular 553. 1955" S. caelestis S. caelestis S. caelestis S. caelestis Agar Medium strain 2221821 NRRL 2418 strain 22218a NRRL 2418 Bennett's S a white c li t gray 263 gm white 264 gm light gray R 2ca light ivory. Zgc bam 0o, chamois 89 gm pale yellow 90 gm grayish eggshell 2ge covert tan, 94m light olive griege brown TABLE 2 ,i entinueq NBS Circular 553, 1955" S. caelestis S. caelestis S. caelestis S. caelestis Agar Medium strain 222l8a NRRL 2418 strain 222l8a NRRL 24l8 Czapeks S a white 263 gm white sucrose R 2ca light ivory, 89 gm pale yellow eggshell W. .7 "PM AW A s a;

Maltose- S a white c light gray 263 gm whise 264 gm light gray tryptone R 2ca light ivory, 3lg adobe brown. 89 pale yeklow 77 gm moderate eggshell cinnamon brown, yellowish brown light brown P 2ge covert tan. 3ig beige brown, 94m light olive 80m grayish yeklowgriege mist brown brown ish brown 109 gm light gray- 95g moderate olive ish olive brown Yeast extract S 2ba pearl, shell c white 92 gm yellowish 264 gm light gray malt extract tint white (lSP-Z) R 2ca light ivory, 2ie light mustard 89 gm pale 91 gm dark grayish eggshell tan yellow yellow 94 g light olive brown 106 g light olive P 2ge covert tan 2ge covert tan, 94 light olive 94 m light olive griege griege brown brown 104 gm light gray- I09 gm light grayish ish olive olive Oatmeal S c light gray e gray 264 gm light gray (lSP-3) R l /ca cream 1% c putty 89 gm pal'e yeklow 90 gm grayish yellow 83 m yellowish gray 1 dc putty, griege 121 m pale yellow green 122 g grayish yellow green lnorganic- S c light gqay l9dc aqua gray 264 gm light gray 149 g ale green sakts starch I90 in fight bluish y (lSP-4) R 2ca light ivory, 2ec biscuit, com, 89 gm pale 90 gm grayish eggshell oatmeal, sand yellow yellow 2 fe covery gray l ec light citron 94 g light olive l2l m pale yellow gray, putty brown green l|2 gm light I22 g grayish yeklow olive gray green Glycerol- S a white a whise 263 gm white 263 gm white as aragine R 2 ca light ivory, 1% ca cream 89 gm pale yellow 89 gm pale yellow (1 P-S) eggshell S Surface R Reverse P Pigment Jacobson, E., WC. Granville, and CE. Foss. 1948. Color Harmony Manual. 3rd Ed. Container Corporation of America. Chicago.

"Kelly, K.L., and DB. Judd. I955. The lSCC-NBS Method of Designating Colors and a Dictionary of Color Names. U.S. Dept. of Comm. Circ.

353, Washington, DC.

Agar Medium TABLE 3 Cultural Characteristics of S. caelestis Cultures S. caelestis strain 222l8a Peptone-iron S Mone to vdry slight trace R Tan Brown P Pale Tan Brown 0 Melanin negative Melanin positive Calcium malate S Trace white Slight blue-white l; Gray-whise Cream 0 Malate solubilized around growth Glucose asparagine 8 Trace whise Fair blue-gray white R Cream Cream p Skim milk S R Pale yellow-tan Tan-brown P Pale yellow-tan Tan O Casein solubilized under Casein not solubilized frowth Tyrosine S Pale blue-gray Blue-gray R Pale yeklow Light brown 5 Pale yellow Light br w Tyrosine solubilized Tyrosine solubilized .ereaasla TABLE aegu aaqii" Cultural Characteristics of S. caelestis Cultures S. caelestis strain 222l8a Xanthine O Other characteristics TABLE 4 S Pale gray R Cream Pale tan P Cream Pale tan 0 Xanthine solubilized around Xanthine not solubilized growth Nutrient starch S Very slight trace blue-white Pale gqay R Cream Cream yellow P Pale yeklow Yellow 0 Starch hydrolyzed Starch hydrolyzed Yeast extract- S Pale blue-white Blue-gray-white Malt extract R Yellow Gray-tan P Pale yeklow Tan Bennett's S White Heavy pale-blue-white R Cream Cream P Yellow-tan Czapeks sucrose S Trace white Good white R Cream white Pale cream p Maltosc-tryptone S White Heavy pale-blue-whise R Cream Pale tan P Pale t[n Peptone-yeast S extract-iron R Colorless Brown (lSP-6) P Brown Tyrosine (lSP-7) S White tinged with blue White with trace blue R Pink-tan Brown P Lightbrown Gelatin Media Plain P Trace brown at surface Pale yellow-tan brown throughout O No liquefaction No liquefaction Nutrient P Yellow Trace brown at surface Yellow-tan throughout 0 Liquefaction A; No liquefaction Broth Media Synthetic nitrate S Colorless surface ring with trace white aerial p 0 Trace colorless bottom Growth throughout growth Flocculent at base Nitrate not reduced to nitrite Nitrate reduced to nitrite Nutrient nitrate S Colorless surface ring Colorless surface ring with whise aerial growth P Pale tan 0 Compact bottom growth Nitrate reduced to nitrite Nitrate not reduced to nitrite Litmus milk S Red-t[n surface ring Red-tan surface ring 0 pH 6.4 Litmus reduced Coagulation positive pH 4.9

S Surface R Reverse P Pigment TAB LE 4 Continued Utilization of Carbon Compounds in the Synthetic Medium of Pridham and Gottlieb* the Synthetic Medium of Pridham and Gottlie b* S. caelcstis S. caclestis S. caelestis S. caelestis strain 222l8a NRRL 2418 strain 222l8a NRRL 2418 Control (--l (*l 5 5 20. lnositol 2. L-Arabinose 22. Phenol 3. Rhamnose 23. Cresol 4. D-Fructose 24. Na Formate 5. D-Galactose 25. Na Oxalate (l 6. D-Glucose 26. Na Tartrate 7. D-Mannosc 60 27. Na Salicylate 8. Maltosc 28. Na Acetate 9. Sucrose 29. Na Citrate [0. Lactose 30. Na Succinatc l l. Cellobiose NW l2. Raffinose l3. Dextrin Good utilization l4. lnulin i 65 Moderate utilization l5. Soluble Starch Doubtful utilization l6. Glycerol No utili/ntion l7. Dulcitol l i Pridham. 'l.(i. and D. (ioitlich. I948. The Utilization of Carbon Compounds h |8 D-Manniml l) some Actinumycetales as an Aid for Species Dcicrminution. J. Bacteriol. l9. D-Sorbitol F191;!

TABLE 5 Utilization of Carbon Compounds in the Synthetic Medium of Shirling and Gottlieb S. caelestis S. caelestis strain 222l8a NRRL 24l8 Plain agar (Negative Control) D-Glucosc (Positive Control) LArabinosc Sucrose lnositol D-Mannitol D-Fructosc Rhamnosc Celluosc iiiiiiii Hiij: iii+ 1+ H Strong utilization Moderate utilization 1 Doubtful utilization No utilization Shirling. E.B.. and D. Goltlieb. I966. Methods for Characterization of Slrcptomyces Species. Int. J. Syst. Bacteriol. l6z3l3-340.

.carbohydrate, and a nitrogen source, for example, an

assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewers yeast with mild solids, soybean meal, cottonseed meal, cornmeal, mild solids, pancreatic digest of casein, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium.

Production of the compounds of the invention can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and 40C., and preferably between about 20 and 32C. Ordinarily, optimum production of the compounds is obtained in about 2 to days. The medium normally remains basic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compounds and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured; it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compounds, as long as it is such that a good growth of the microorganism is obtained.

The new compounds of the invention, N-demethyl-7- O-demethylcelesticetin (IV) and N-dimethylcelesticetin (IVa), exist either in the non-protonated (free base) form or the protonated (salt) form depending on the pH of the environment. They form stable protonates (acid addition salts) by neutralization of the free base with an acid or by metathesis between the protonated form of the antibiotic and the anion of an acid. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, malcic, fumaric, pamoic, cholic, palmitic, mucic, camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, 3-phenylsalicylic, 5-phenylsalicylic, 3- methylglutaric, orthosulfobenzoic, cyclohexanesulfamic, cyclopentanepropionic, l ,2- cyclohexanedicarboxylic, 4-cyclohexenecarboxylic, octadecenylsuccinic, octenylsuccinic, methanesulfonic, benzenesulfonic, helianthic, Reineckes, azobenzenesulfonic, octadecylsulfuric, picric, and the like acids. These acid addition salts are useful in upgrading the free base.

Because of the presence of the salicylic acid moiety, N-demethyl-7-0-demethylcelesticetin (IV) and N-demethylcelesticetin (lVa) form salts with alkali metals and alkaline earth metals by procedures well known in the art. Salts which can be made are the sodium, potassium, calcium, lithium, and the like. These salts have the uses disclosed above for the acid addition salts.

N-Demethyl-7-0-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa) are soluble in lower alcohols of from one to four carbon atoms, inclusive, and ketones; they are less soluble in water and chlorinated hydrocarbon solvents; and essentially insoluble in ether and saturated hydrocarbon solvents.

A variety of procedures can be employed in the isolalation and purification of N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (N21), for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, and crystallization from solvents.

In a preferred recovery process, N-demethyl-7-O- .demethylcelesticetin (IV) and N-demethylcelesticetin (IVa) are recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotics are then removed from the filtered or centrifuged broth by resin sorption on a resin comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene. Suitable resins are Amberlite XAD-l and XAD-Z disclosed in US. Pat. No. 3,515,717. The resin is eluted with an organic or aqueous organic solvent in which the sorbed antibiotics are soluble. percent aqueous methanol is the preferred solvent for elution. The antibiotic mixture in the methanol eluates is purified by concentrating the eluates to dryness and then subjecting them to counter double current distribution using l-butanol-water 1:1 v/v) as the solvent system. Fractions containing N- demethyl-7-0-demethylcelesticetin are concentrated to dryness and the antibiotic crystallizes by triturating with methanol.

Fractions containing N-demethylcelesticetin are sub- .jected to countercurrent distribution using a solvent system consisting of l-butanol-water [:1 v/v). The N- fords a facile separation of the antibiotics. Fractions,

from the silica gel chromatography column which contain only N-demethylcelesticetin are concentrated to dryness; the resulting residue is dissolved in methanolic hydrogen chloride, and this solution is mixed with ether. The resulting precipitated N-demethylcelesticetin hydrochloride is isolated by filtration and dried.

The presence of N-demethyl-7-0-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa) in the fermentation beer and various recovery liquids is determined by activity against the microorganism S. lutea. This is a standard microbiological disc plate assay using 12.5 mm. discs. Thin layer chromatography (tlc) using silica gel G and a solvent mixture consisting of chloroform-methanol (6:1 v/v) is used to identify the presence of either or both of the antibiotics in a sample showing activity against S. lutea, as described above. On this chromatographic system, N-demethyl-7-0- demethylcelesticetin (IV) has an Rf of 0.05 and N-demethylcelesticetin (IVa) has an Rf of 0.15.

Alkaline hydrolysis of N-demethyl-7-0-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa) with a suitable base, for example, sodium hydroxide, for 16 hours at room temperature results in the cleavage of the ester group and the formation of N- demethyl-7-O-demethyldesalicetin (V) and N-demethylldesalicetin (Va), respectively. Other bases, for example, aqueous ammonium hydroxide and organic amines can be used instead of sodium hydroxide. The reaction can be carried out in solvents like lower alcohols (methanol, ethanol) by refluxing in the presence of base. Duration of the treatment depends on the strength of the base and the temperature used.

. Salicylic acid, which is formed as a by-product during the above reaction, can be removed by extraction with e he aLBHiQLm I-Iydrazinolysis of N-demethyl-7-O-demethylcelesticetin (IV) with hydrazine hydrate at reflux for about 23 hours affords proline hydrazide which can be transformed to proline hydrochloride by heating with aqueous hydrochloric acid. The second product of the above hydrazinolysis reaction is B-hydroxyethylthiolinqesamittideKBfiELUYJ). W

N-Demethyl-7-0-demethyldesalicetin (V) and N-demethyldesalicetin (Va) exist either in the nonprotonated (free base) form or the protonated (salt) form depending on the pH of the environment. Acid addition salts of these compounds can be made as disclosed for the N-demethyl-7-0-demethylcelesticetin tlvland.N:slcmstbxls leakaiutlyal.--

N-Demethyl-7-0-demethylcelesticetin (IV) and N-demethylcelesticetin (Na) and their salts are active against Staphylococcus aureus and Streptococcus faecalis and can be used to disinfect washed and stacked food utensils contaminatd with these bacteria; they can also be used as disinfectants on various dental and medical equipment contaminated with Staphylococcus aureus. Further, since these antibiotics are active against Sreptococcus hemolyticus, they can be used to disinfect instruments, utensils or surfaces where the inactivation of this organism is desirable.

N-Demethyl-7-0-demethyldesalicetin (V) and N-d methylslss liset n We), a d hawk e qes s against Staphylococcus aureus and can be used for the same purposes given for N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa).

N-Demethyl-7-0-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa) can be converted to various antibacterially active analogues as represented by structural formulae VII, Vlla, VIII, and VIIIa. In the following discussion, in cases where acylation, phosphorylation, or 7epimerization via 7-oxidation of compounds, IV, lVa, V, or Va are involved, the compound being reacted should be suitably N-protected according to the art, for example as the N-carbobenzoxy derivative. The carbobenzoxy group is removed after reaction or at a suitable later time by hydrogenolysis, according to the art. For example, N-demethyl-7-epi-7-0-demethylcelesticetin can be prepared by the procedure disclosed in U.S. Pat. No. 3,514,440, column 2, line 44 to column 3, line 10; N-demethyl-7(R)- and 7(S)-halo-7- demethoxycelesticetin can be prepared by the methods disclosed in U.S. Pat. No. 3,496,163, using the modifled Rydon procedure as given in Example 6 of said patent; N-demethyl-7(R)-0-alkyl-7-0-demethylcelesticetin can be prepared by esterification of N- demethyl-7(R)-0-alkyl-7-0-demethyldesalicetin at the primary hydroxyl with salicyloyl chloride as disclosed in U.S. Pat. No. 2,851,463, especially column 2, lines et seq.; N-demethyl-7(S)-0-alkyl-7-0-demethylcelesticetin can be prepared from N-demethyl-7(S)-O- alkyl-7-0-demethyldesalicetin by the procedure disclosed above for the preparation of the 7(R) isomer; 2-0-phosphates can be prepared by the procedures closed in U.S. Pat. No. 3,487,O6 8 2-0-acy|ates can be prepared by the acylation procedures disclosed in U.S. Pat. No. 3,326,891 and U.S. Pat. No. 3,426,012; and N-alkyl compounds of formulae VII and Vlla can be prepared by procedures disclosed in U.S. Pat. No. 3,380,992 with the provision that the starting compound of formula V in U.S. Pat. No. 3,380,992 is to be the present compound of formula IV or Na in the subject invention; i.e., wherein Y of formula V in U.S. Pat. No. 3,380,992 is a-SCH2CH R is Cl-I and R, R and R are H for IV, and R, and R are CH R and R are H for Na. The most pertinent portions of U.S. Pat. No. 3,380,992 are column 2, lines 17 et seq., column 13, lines 43-69, and Example 1, parts F-2 to F-4 and 6-1, column 19, line 74 to column 20, line 52.

N-I-Iydroxyalkyl compounds for formulae VII and VlIa can be prepared by reacting the compound of formula IV or lVa, advantageously as the hydrochloride acid addition salt, with ethylene oxide, propylene oxide, butylene oxide or pentylene'oxide, preferably in a pressure vessel in which the reaction proceeds as follows as illustrated with ethylene oxide:

The reaction mixture after cooling is worked up in the usual manner using such procedures as distillation, solvent extraction, crystallization and the like.

The proportions are not critical but an excess of alkylene oxide is desirable. An excess of from 2 to 100 times the stoichiometric amount is suitable. The temperature also is not critical. At temperatures below about 35C., however, the reaction is undesirably slow and ordinarily it will not be necessary or desirable to exceed about 200C. The reaction proceeds well at 100C.

Advantageously the reaction is carried out in an inert mutual solvent for the reactants. Suitable such solvents include methyl alcohol, ethyl alcohol, propyl alcohol, benzene, toluene, cyclohexane, and tetrahydrofuran.

Alternatively the alkylation can be effected by a reductive alkylation such as described in US. Pat. No.

3,496,163, Part Gl-4, by substituting the formaldehyde (formalin) by hydroxyacetaldehyde, 3-hydroxypropionaldehyde, 4-hydroxybutyraldehyde or 5- hydroxypentaldehyde.

Salts of the compounds of structural formulae VII and VIla can be made as disclosed for compounds IV and IVa. These salts are useful in the same manner as the non-protonated compounds, and further, they can be used to upgrade the non-protonated compounds by procedures well known in the art.

N-Demethyl-7-0-demethyldesalicetin (V) and N-memethyldesalicetin (Va) can be converted to various antibacterially active analogues as represented by structural formulae VIII and VIIla. For example, N- demethyl-7-epi-7-0-demethyldesalicetin can be prepared by hydrolysis of the B-O-salicylyl acyl of N- demethyl-7-epi-7-0-demethylcelesticetin methods disclosed in US. Pat. No. 2,851,463, especially column 1, line 27 to column 2, line 56 [N-demethyl7-epi-7-0- demethylcelesticetin can be prepared by methods disclosed in US. Pat. No. 3,514,440, as noted above]; B-acylates of N-demethyl-7-epi7-0-demethyldesalicetin and N-demethyldesalicetin can be prepared by the procedures disclosed in US. Pat. No. 2,851,463, particularly those disclosed in column 2, line 56 to column 3, line 16; N-demethyl-7(R)- and -7(S)-halo-7-demethoxydesalicetin can be prepared by hydrolysis of N- demethyl-7(R)- and -7(S)-halo-7-demethoxycelesticetin by methods disclosed in US. Pat. No. 2,851,463, especially column 1, line 27 to column 2, line 56, except that the process should be carried out at room temperature, at a pH of 8 10, preferably about pH 10, so as to selectively remove the salicyloyl and not hydrolyze off the 7-halo group [N-demethyl7(R)- and -7(S)- halo -7-demethoxycelesticetin can be prepared by methods disclosed in US. Pat. No. 3,496,163, as noted above]; N-demethyl-7(R)-0-alkyl-7-0-demethyldesalicetin can be prepared by reacting N-demethyl-7(R)0- alkyl-HETL [this compound can be prepared by substituting HETL (V1) for the compound methyl thiolincosaminide (MT L) in the procedures disclosed in US. Pat. No. 3,574,187] with ethyl chloroformate according to methods known in the art to give the N-demethyl-7(R)-0-alkyl-I-IETL B-O-ethylcarbonate ester which then can be N-acylated with N-carbobenzoxy-L-proline by the procedures disclosed in US. Pat.

No. 3,380,992, especially Example 6-the ethyl car bonate ester protective group can be removed by treatment with a mild base. eg. NaHCO N-demethyl-7(S)- O-alkyl-7-0-demethyldesalicetin can be prepared from N-demethyl-7(S)-0-alkyl-7-O-demethyl-HETL according to the procedure given above for the 7(R) isomer; 2-O-phosphates can be prepared by first preparing the ,B-O-(alkyl carbonate)-ester of N-demethyl-7-0-demethyldesalicetin (V) or N-demethyldesalicetin (Va) by reaction of the desalicetin compound with an alkyl chloroformate using procedures known in the art, and then phosphorylating the reaction product according to the procedures disclosed in U.S. Pat. No. 3,487,068the alkyl carbonate protective group can be removed by treatment of the phosphorylated product with mild base, e.g., NaHCO B-O-acylates can be prepared by the procedures disclosed in US. Pat. No. 2,851,463, especially column 2, lines et seq., using one mole of acylating agent per mole of desalicetin compound; 2-0-acylates can be prepared by the acylation procedures disclosed in US. Pat. No. 3,326,891 and US. Pat. 'No. 3,426,012; N-alkyl and N- hydroxyalkyl compounds of formulae VIII and Vllla can be prepared as described above for similar derivatives of formulae VII and VIIa.

Acid addition salts of the compounds of structural formulae VIII and VIIla can be made as disclosed for compounds IV and Na. These salts are useful in the same manner as the non-protonated compounds, and, further, they can be used to upgrade the nonprotonated compounds by procedures well known in the art.

The following examples are illustrative of the process and products of the present invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 N-DEMETHYL-7-0-DEMETHYLCELES- TICETIN (IV) Part A.

Fermentation Mycelium and/or spores from a slant of Streptomyces caelestis strain 2218a, NRRL 5481, are used to inoculate a series of 500-ml. Erlenmeyer flasks each containing ml. of seed medium consisting of the following ingredients:

Glucose monohydrate 25 gm./l. Pharmamedia* 25 gm./l, Tap water q.s. l liter Pharmamedia is an industrial grade of cottonseed flour produced by 'l'radcr's Oil Cu, Ft. Worth, Texas.

The presterilization pH of the seed medium is 7.2. The seed is grown for three days at 28C. on a Gump rotary shaker operating at 250 r.p.m.

The fermentation proper is carried out in the medium of the following composition:

Glucose monohydrate 25 gm./l. Wilson's Peptone Liquor No. 159* 15 gm./l. Yeast extract 2.5 gm./l. Tap water q.s. 1 liter Wilson's Peptone Liquor No. 159 is a preparation of enzymatically hydrolyzed proteins of animal origin.

The medium is adjusted to pH 7.2 7.4 with 50 percent sodium hydroxide, distributed in 100 ml. volumes/500 ml. Erlenmeyer flasks and sterilized at C. for 30 minutes. The inoculated flasks (5 percent inoculum) are incubated at 28C. on a Gump rotary shaker operating at 250 rpm. Optimum production of N- demethyl-7-O-deniethyleelesticetin (IV) and N-demethylcelesticetin (lVa) is obtained after about four days qf er s on- Ihqtitsrq thqssant lz sztiqsi h inentation beer is determined by the activity of the antibiotics against the microorganism S. lutea by a standard microbiological disc plate assay using 12.5 mm. discs.

A scaled-up fermentation process is as follows:

Shake Flask Pre-seed Medium Glucose monohydrate l gm./l. \Vilsons Peptone Liquor No. 159 gmJl. Cornsteep liquor l0 gm./l. .Pharmamedia 2 gm./l. Tap water q.s. l liter ces caelestis strain 222l8a, NRRL 5,481 and the seed flasks are then grown for three days at 28C. on a -Gump rotary shaker operating at 250 rpm. Three shake flasks (300 ml.) of the pre-seed inoculum, described above, are used to inoculate a 300-liter seed tank containing 250 liters of the seed medium described above for the seed flasks with the addition of Ucon (a polyalkylene glycol synthetic defoaming fluid supplied by Union Carbide Chemical Co.) as anantifoaming agent. The seed tank is maintained at a temperature of 28C. for 48 hours with agitation (280 rpm.) and air flow of 100 SCFM (standard cubic feet/minute) at psig.

The seed tank, described above, is used to inoculate a 7,000-liter fermentation tank containing 5,000 liters of the following sterile medium:

Glucose monohydrate l5 gmJl. Wilsons Peptone Liquor No. I59 l5 gm./l. Yeast extract l gm./l.

Ucon is added as an antifoaming agent. The fermentation tank is inoculated at the rate of 5 percent seed and maintained at 28C. for four days with an air flow of SO SCFM at 10 psig and agitation at a rate of 166 r.p.m.

Part B.

Recovery Whole fermentation beer (approximately 4,400 liters), obtained as described above, isftltered using diatomaceous earth as a filter aid. The filter cake is washed with water. The wash is combined with the clear beer and the combined solution is passed through 150 liters of Amberlite XAD-2 resin at a flow rate of 150 liters per hour. The spent beer is discarded. The column is washed with water (75 liters) and then eluted with 1100 liters of 95 percent aqueous methanol. The methanolic eluates are collected in two pools: Pool 1 (4301.) is concentrated to dryness; yield, 1,275 g. Pool II (970 l.) is also concentrated to dryness; yield 343 g.

Part C.

Isolation of N-Demethyl-7-0-Demethylcelesticetin Hydrochloride by Counter Double Current Distribution A portion of Pool I, described'above, is subjected to purification by counter double current distribution using a solvent system consisting of l-butanol-water 51) t o a nstystal insf N-q me h -dsme ylcelesticetin hydrochloride. 50 G. of the material obtained from Pool I is dissolved in both phases (200 ml. of each phase) of the above-noted solvent system and the pH is adjusted to 3.5 with 2 N aqueous hydrochloric acid. The solution is put in tubes 12 at the point where the lower phase enters the countercurrent distribution machine. The following transfers are run:

1. 30 transfers without collecting fractions.

2. 32 transfers with collection of the upper phas only.

3. 50 transfers collecting both upper and lower phases. The distribution is analyzed by determination of bioactivity of selected fractions against S. lutea.

Lower Collector l5 traces 20 l l 12.5 I4 14.5 I6 I7 Lower Machine lemmin s 5 l 3 I0 I I I5 I l 20 I0 25 traces 3O traces 35 traces 40 I5 45 traces 50 0 22Qt CQl 82 0 75 l 3 70 l 2.5 65 0 60 0 0 50 l l 45 0 40 0 35 0 30 traces 25 10 20 l2 l5 l7 10 22.5

Selected fractions are pooled. Each pool is concentrated to dryness in vacuo to give the following preparations respectively.

Pool AI, lower collector 3050; lower machine 50-46. Pool BI, lower machine 37-0. Pool CI, upper collector l5-0. Pool AI contains traces of bio-activity and is discarded. Pool BI is dissolved in a minimum amount of methanol and the solutions are mixed with 200 ml. of ether. The resulting precipitated material is collected by filtration and dried; yield, 2. l3 g., labelled Example 2 N-DEMETHYLCELESTICETIN (lVa) The filtrate from the trituration with methanol of the five preparations obtained from Pool Bll, as disclosed in Example 1, is added to two liters of ether. The resulting precipitated material is isolated by filtration and dried; yield, l0.63 g., labelled Bll-l. This material is combined with preparation labelled Bl-l, obtained as disclosed in Example 1, to give 12.7 g. of material containing N-demethylcelesticetin ([Va), and labelled BH- 2. This material is then subjected to counter current distribution using the solvent system l-butanol-water (1:1 v/v). The starting material (12.7 g. of preparation Bil-2) is dissolved in both phases of the above-noted solvent system and added into five tubes of a 500-tube all glass counter current distrition apparatus. The distribution is analyzed for antibiotic content by using Sarcina lutea as the test organism.

After 1,500 transfers, tubes 340-400 are combined and the solution concentrated to dryness. The residue is dissolved in 50 ml. of methanol and this solution is mixed with one liter of ether to give a precipitate; yield, 1.5 g. labelled Bil-3. This material is found by tlc chromatography (silica gel G, chloroform-methanol 6:1 v/v) to be a mixture of N-demethylcelesticetin (Na) and N-demethyl-7-0-demethylcelesticetin (IV). These two antibiotics are separated by silica gel chromatography as follows: The silica gel chromatography column is prepared from 250 g. of silica gel G (Merck- Darrnstadt ART. 7734) packed in the solvent system consisting of chloroform-methanol (6:1 v/v). The starting material (1.3 g. of Bil-3) is dissolved in the solvent and the solution is mixed with 20 g. of silica gel. The mixture is concentrated to dryness and the obtained powder is added on the top of the column bed. The column is eluted with the above solvent system. Fractions (20 ml. each) are analyzed by bio-activity against Sarcina lutea and ultraviolet determinations. Results follow:

Fraction No. Zone (S. lutea, mm.) UV (A max. OD)

-Continued Fraction No. Zone (5. lutea. mm.) UV (A max. OD)

300 I9 305 (0.23) 3l0 17.5 305 (0.20) 320 17.5 305 (0.24) 330 16 3H) (0.40)

350 13 Non descriptive UV 400 I2 Non descriptive UV 500 I2 Non descriptive UV 600 traces Non descriptive UV Fractions l60240 are combined and the solution is concentrated to dryness. The residue is dissolved in l N methanolic hydrogen chloride 10 ml.) and this solution is mixed with ether. The resulting precipitated material is isolated by filtration and dried; yield, l30 mg. of N-demethylcelesticetin hydrochloride (Vla).

Example 3 N-DEMETHYL-7-0-DEMETHYLDESALICETIN (V) Upon the treatment of N-demethyl-7-0-demethylcelesticetin (IV) with 50 percent aqueous NaOH for about 16 hours at room temperature, there is produced N-demethyl-7-0-demethyldesalicetin (V). Salicylic acid which is fomled as a by-product during the reaction can be removed by extraction with ether at pH 3.0.

Example 4 'N-DEMETHYLDESALICETIN v.1)

Upon the treatment of N-demethylcelesticetin (lVa) with 50 percent aqueous NaOH for about 16 hours at room temperature. there is produced N-demethyldesalicetin (Va). Salicylic acid which is formed as a byproduct during the reaction can be removed by extraction with ether at pH 3.0.

Example 5 fi-HYDROXYETHYLTHlOLINCOSAMlNlDE (B-HETL) (VI) Upon hydrazinolysis of N-demethyl-7-O-demethylcelesticetin (IV) with hydrazine hydrate at reflux for about 23 hours, there is produced 'n-HETL, proline hydrazide and salicylic acid hydrazide. BHETL is recovered from the reaction mixture by counter current distribution in the solvent system consisting of equal volumes of l-butanol and water. This procedure separates B-HETL from proline hydrazide and salicylic acid hydrazide.

We claim: 1. A process for preparing the antibiotic N-demethyl- 7-0-dernethylcelesticetin having the structural formula:

HO'CH H I N OH h and the antibiotic N-demethylcelesticetin having the structural formula:

H all a H P N custo al I SCHz menu-Q 3. A process according to claim 1 wherein said produced antibiotics N-demethyl-7-0-demethylcelesticetin and N-demethylcelesticetin are isolated from the fermentation medium by a process which comprises:

a. filtering the fermentation whole broth to obtain filtered beer;

b. passing the filtered beer over a resin comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene; and,

c. eluting said resin with a solvent selected from the group of organic or aqueous organic solvents in which N-demethyl-7-0-demethylcelesticetin and N-demethylcelesticetin are soluble to obtain eluates containing said antibiotics.

4. A process for isolating N-demethyl-7-()-demethylcelesticetin from a mixture as defined in claim 3, which comprises subjecting said mixture to counter double current distribution using a solvent system consisting of l-butanol-water 1:1 v/v 5. A process for isolating N-dcmcthylcclcsticctin from a mixture as defined in claim 3 which comprises subjecting said mixture to counter current distribution using a solvent system consisting of l-butanol-water 1:1 v/v), isolating active fractions and subjecting them to silica gel chromatography using a solvent system consisting of chloroform-methanol (61] v/v) to isolate N-demethylcelesticetin substantially free of N- demethyl-7-O-demethylcelesticetin. =l l UNITED STATES PATENT OFFICE CERTIFICATE CF CORRECTION Page 1 Patent No. 5,847,7 l6 Dated November 12, 197% Inventor) Alexander D Argoudel is, John H Coats, Oldr ich It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Ergnt gage, under Inventors should include Oldrich K. Sebek, Kalamazoo. Column l, l in-e A8, for "demethyldesal icetain read demethyldesal icetin Column 6, l lne 8, For hycrocarbn" read hydrocarbon Column 9, l ine 57, for "Taable" read Tab e TABLE 2: Column ll, under Agar Medium, for "Inorganic sakts starch" read Inorganic salts starch under S. caelestis strain 22218a, for "c l ight gqay read c l ight gray under S. caelestis NRRL 2 l18, for a whise" read a white Column 12, under 5-. caelestis strain 22218a, For "265 gm whise" read 265 gm white for 89 ale yeklow" read 89 pale yellow for "89' gm pale el low' read 89 gm pale yellow under S. caelestis NRRL 2 i1 for "m grayish yeklowish brown" read 80 m grayish yellowish brown For m yellowish gray" read --95 m yel low ish gray For "122 g gray ish yeklow read 122 g grayish yel low Column 10, under S. caelestis NRRL 2 l18, For gm grayish read 90 gm grayish yellow TABLE 2: Titles should be set out l ike this Agar Medium S. caelestis S. caelestis stra in 22218a NRRL 2418 Column 11, under S. caelestis strain 22218a, For "Gray-whise" read Gray-white for "Trace-whise read Trace-white for "Casein solubil ized under Frowth" read--Casein solubil ized under growth under S. caelestis NRRL 2e18, for "Mone to vdry' read None to very Under Column 11, S. caelestis strain 22218a, for "Pale yeklow read Pale yellow Column 15, under S. caelestis strain 22218a, for Pale yeklow" read Pale yellow; again for Pale yeklow" read Pale yel low For "Red-t[n surface ring read Red-tan surface ring under S. caelestis NRRL 2 l18, For "Pale gqay" read Pale gray for "Heavy ale-blue-whise read Heavy paleblue-white for "Pale t[n read Pale tan for F ORM PO-OSO (10-69) USCOMM'DC 50376P59 ".5. GOVERNMENT PRINTING OFFICE (9G8 0-880-334 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Page 2 Patent No. 5, 7,7 Dated November 12, 1974 Alexander D. Argou'del is, John H. Coats, Oldrich K. Sebek Inventofls) It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

"Colorless surface r ing with wh ise aer ial growth" read Colorless surface r ing with white aer ial growth Column 15, l ine 55, for "mild sol ids read milk sol ids l ine 56, for "m i ld sol ids" read mil k sol ids ".1

Column 19, l ine 26, for "N-memethyldesal icetin" read N-demethyldesal icetin Column 20, l ine 37, for "2218a" read 22218a Column 22, l ine 20, under title column Zone (mm) of inhibition, read 15 Column 25,

l ine 7, for HETL read B-HETL Signed and sealed this 24th day of June 1975.

(SEAL) fattest:

C. MARSHALL DAN-N RUTH C. Iii-130K Commissioner of Patents Attesting Officer and Trademarks Foam Po-wso (IO-69) USCOMM-DC 50376-P69 i ".5, GOVIRIIIIENT PRINTING OFFICE "I. O-QOl-IM 

1. A PROCESS FOR PREPARING THE ANTIBIOTIC N-DIMETHYL-7-0DEMETHYLCELESTICETIN HAVING THE STRUCTURAL FORMULA:
 2. A process according to claim 1 wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.
 3. A process according to claim 1 wherein said produced antibiotics N-demethyl-7-0-demethylcelesticetin and N-demethylcelesticetin are isolated from the fermentation medium by a process which comprises: a. filtering the fermentation whole broth to obtain filtered beer; b. passing the filtered beer over a resin comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene; and, c. eluting said resin with a solvent selected from the group of organic or aqueous organic solvents in which N-demethyl-7-0-demethylcelesticetin and N-demethylcelesticetin are soluble to obtain eluates containing said antibiotics.
 4. A process for isolating N-demethyl-7-0-demethylcelesticetin from a mixture as defined in claim 3, which comprises subjecting said mixture to counter double current distribution using a solvent system consisting of 1-BUTANOL-WATER (1:1 v/v).
 5. A process for isolating N-demethylcelesticetin from a mixture as defined in claim 3 which comprises subjecting said mixture to counter current distribution using a solvent system consisting of 1-butanol-water (1:1 v/v), isolating active fractions and subjecting them to silica gel chromatography using a solvent system consisting of chloroform-methanol (6:1 v/v) to isolate N-demethylcelesticetin substantially free of N-demethyl-7-0-demethylcelesticetin. 